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A series of dihydropyridinone (DHP) compounds was prepared and evaluated for MGAT2 activity. The efforts led to the identification of novel tetrazolones with potent MGAT2 inhibitory activity and favorable in vitro profiles. Further tests of select analogues in mouse models revealed significant reduction in food intake and body weight. Subsequent studies in MGAT2 knockout mice with the lead candidate 12 (BMS-986172) showed on-target- and mechanism-based pharmacology. Moreover, its favorable pharmacokinetic (PK) profile and the lack of species variability in the glucuronidation potential resulted in a greater confidence level in the projection of a low dose for achieving targeted efficacious exposures in humans. Consistent with these projections, PK data from a phase 1 trial confirmed that targeted efficacious exposures could be achieved at a low dose in humans, which supported compound 12 as our second and potentially superior development candidate for the treatment of various metabolic disorders.

Inhibition of monoacylglycerol transferase 2 (MGAT2) has recently emerged as a potential therapeutic strategy for the treatment of metabolic diseases such as obesity, diabetes and non-alcoholic steatohepatitis (NASH). Metabolism studies with our clinical lead (1) suggested variability in in vitro glucuronidation rates in liver microsomes across species, which made projection of human doses challenging. In addition, the observation of deconjugation of the C3-C4 double bond in the dihydropyridinone ring of 1 in solution had the potential to complicate its clinical development. This report describes our lead optimization efforts in a novel pyridinone series, exemplified by compound 33, which successfully addressed both of these potential issues., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

MGAT2 inhibition is a potential therapeutic approach for the treatment of metabolic disorders. High-throughput screening of the BMS internal compound collection identified the aryl dihydropyridinone compound 1 (hMGAT2 IC 50 = 175 nM) as a hit. Compound 1 had moderate potency against human MGAT2, was inactive vs mouse MGAT2 and had poor microsomal metabolic stability. A novel chemistry route was developed to synthesize aryl dihydropyridinone analogs to explore structure-activity relationship around this hit, leading to the discovery of potent and selective MGAT2 inhibitors 21f , 21s , and 28e that are stable to liver microsomal metabolism. After triaging out 21f due to its inferior in vivo potency, pharmacokinetics, and structure-based liabilities and tetrazole 28e due to its inferior channel liability profile, 21s (BMS-963272) was selected as the clinical candidate following demonstration of on-target weight loss efficacy in the diet-induced obese mouse model and an acceptable safety and tolerability profile in multiple preclinical species.

Pain medication plays an important role in the treatment of acute and chronic pain conditions, but some drugs, opioids in particular, have been overprescribed or prescribed without adequate safeguards, leading to an alarming rise in medication-related overdose deaths. The NIH Helping to End Addiction Long-term (HEAL) Initiative is a trans-agency effort to provide scientific solutions to stem the opioid crisis. One component of the initiative is to support biomarker discovery and rigorous validation in collaboration with industry leaders to accelerate high-quality clinical research into neurotherapeutics and pain. The use of objective biomarkers and clinical trial end points throughout the drug discovery and development process is crucial to help define pathophysiological subsets of pain, evaluate target engagement of new drugs and predict the analgesic efficacy of new drugs. In 2018, the NIH-led Discovery and Validation of Biomarkers to Develop Non-Addictive Therapeutics for Pain workshop convened scientific leaders from academia, industry, government and patient advocacy groups to discuss progress, challenges, gaps and ideas to facilitate the development of biomarkers and end points for pain. The outcomes of this workshop are outlined in this Consensus Statement.

The potent MCHR1 in vitro and in vivo antagonist activity of a series of cyclic tertiary alcohols derived from compound 2b is described. Subsequent pharmacokinetic and pharmacodynamic studies identified BMS-814580 (compound 10) as a highly efficacious antiobesity agent with a relatively clean in vitro and in vivo safety profile.

Non-basic azolotriazinones were explored using an empirical free brain exposures-driven approach to identify potent MCHR1 antagonists for evaluation in in vivo efficacy studies. An optimized lead from this series, 1j (rMCHR1 Ki=1.8 nM), demonstrated a 6.9% reduction in weight gain relative to vehicle in a rat model at 30 mg/kg after 4 days of once-daily oral treatment as a glycine prodrug. Despite a promising efficacy profile, an assessment of the biliary toxicity risk of this compound rendered this compound non-progressible., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Our investigation of the structure-activity and structure-liability relationships for dihydropyrrolopyrazol-6-one MCHR1 antagonists revealed that off-rate characteristics, inferred from potencies in a FLIPR assay following a 2 h incubation, can impact in vivo efficacy. The in vitro and exposure profiles of dihydropyrrolopyrazol-6-ones 1b and 1e were comparable to that of the thienopyrimidinone counterparts 41 and 43 except for a much faster MCHR1 apparent off-rate. The greatly diminished dihydropyrrolopyrazol-6-one anti-obesity response may be the consequence of this rapid off-rate., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Identification of MCHR1 antagonists with a preclinical safety profile to support clinical evaluation as antiobesity agents has been a challenge. Our finding that a basic moiety is not required for MCHR1 antagonists to achieve high affinity allowed us to explore structures less prone to off-target activities such as hERG inhibition. We report the SAR evolution of hydroxylated thienopyrimidinone ethers culminating in the identification of 27 (BMS-819881), which entered obesity clinical trials as the phosphate ester prodrug 35 (BMS-830216).

Several strategies have been employed to reduce the long in vivo half-life of our lead CB1 antagonist, triazolopyridazinone 3, to differentiate the pharmacokinetic profile versus the lead clinical compounds. An in vitro and in vivo clearance data set revealed a lack of correlation; however, when compounds with <5% free fraction were excluded, a more predictable correlation was observed. Compounds with log P between 3 and 4 were likely to have significant free fraction, so we designed compounds in this range to give more predictable clearance values. This strategy produced compounds with desirable in vivo half-lives, ultimately leading to the discovery of compound 46. The progression of compound 46 was halted due to the contemporaneous marketing and clinical withdrawal of other centrally acting CB1 antagonists; however, the design strategy successfully delivered a potent CB1 antagonist with the desired pharmacokinetic properties and a clean off-target profile.

The 5-HT2C receptor has been implicated as a critical regulator of appetite. Small molecule activation of the 5-HT2C receptor has been shown to affect food intake and regulate body weight gain in rodent models and more recently in human clinical trials. Therefore, 5-HT2C is a well validated target for anti-obesity therapy. The synthesis and structure-activity relationships of a series of novel tetrahydropyrazinoisoquinolinone 5-HT2C receptor agonists are presented. Several members of this series were identified as potent 5-HT2C receptor agonists with high functional selectivity against the 5-HT2A and 5-HT2B receptors and reduced food intake in an acute rat feeding model upon oral dosing., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A series of 2,3,3a,4-tetrahydro-1H-pyrrolo[3,4-c]isoquinolin-5(9bH)-ones is described, several examples of which exhibit potent 5-HT(2C) agonism with excellent selectivity over the closely related 5-HT(2A) and 5-HT(2B) receptors. Compounds such as 38 and 44 were shown to be effective in reducing food intake in an acute rat feeding model., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dapagliflozin is a potent and selective sodium glucose cotransporter-2 (SGLT2) inhibitor which promotes urinary glucose excretion and induces weight loss. Since metabolic compensation can offset a negative energy balance, we explored the potential for a compensatory physiological response to the weight loss induced by dapagliflozin. Dapagliflozin was administered (0.5-5 mpk; p.o.) to diet-induced obese (DIO) rats with or without ad libitum access to food for 38 days. Along with inducing urinary glucose excretion, chronic administration of dapagliflozin dose-dependently increased food and water intake relative to vehicle-treated controls. Despite this, it reduced body weight by 4% (relative to controls) at the highest dose. The degree of weight loss was increased by an additional 9% if hyperphagia was prevented by restricting food intake to that of vehicle controls. Neither oxygen consumption (vO2) or the respiratory exchange ratio (RER) were altered by dapagliflozin treatment alone. Animals treated with dapagliflozin and pair-fed to vehicle controls (5 mpk PF-V) showed a reduction in RER and an elevation in nonfasting β-hydroxybutyrate (BHBA) relative to ad libitum-fed 5 mpk counterparts. Fasting BHBA was elevated in the 1 mpk, 5 mpk, and 5 mpk PF-V groups. Serum glucose was reduced in the fasted, but not the unfasted state. Insulin was reduced in the non-fasted state. These data suggest that in rodents, the persistent urinary glucose excretion induced by dapagliflozin was accompanied by compensatory hyperphagia, which attenuated the weight loss induced by SGLT2 inhibition. Therefore, it is possible that dapagliflozin-induced weight loss could be enhanced with dietary intervention.

Aim: To test the antidiabetic efficacy of ibipinabant, this new cannabinoid receptor 1 (CB1) antagonist was compared with food-restriction-induced weight loss, rosiglitazone (4 mg/kg) and rimonabant (3 and 10 mg/kg), using parameters of glycaemic control in male Zucker diabetic fatty (ZDF) rats., Methods: Body weight, food and water intake, fasted and non-fasted glucose and insulin, glucose tolerance and glycosylated haemoglobin (HbA1c) were all assessed over the course of the 9-week study. Pancreatic insulin content and islet area were also evaluated., Results: At the end of the study, vehicle-treated ZDF rats were severely hyperglycaemic and showed signs of β-cell decline, including dramatic reductions in unfasted insulin levels. Ibipinanbant (10 mg/kg) reduced the following relative to vehicle controls: fasting glucose (-61%), glucose excursion area under the curve (AUC) in an oral glucose tolerance test (OGTT, -44%) and HbA1c (-50%). Furthermore, non-fasting insulin, islet area and islet insulin content were all increased (71, 40 and 76%, respectively) relative to vehicle controls by the end of the study. All of these effects were similar to those of rimonabant and rosiglitazone, where ibipinabant was slightly more effective than rimonabant at the lowest dose and somewhat less effective than rosiglitazone at all doses. These antidiabetic effects appear independent of weight loss because none of the parameters above were consistently improved by the comparable weight loss induced by food restriction., Conclusions: Ibipinabant may have weight loss-independent antidiabetic effects and may have the potential to attenuate β-cell loss in a model of progressive β-cell dysfunction., (© 2012 Blackwell Publishing Ltd.)

Obesity remains a significant public health issue leading to Type II diabetes and cardiovascular disease. CB1 antagonists have been shown to suppress appetite and reduce body weight in animal models as well as in humans. Evaluation of pre-clinical CB1 antagonists to establish relationships between in vitro affinity and in vivo efficacy parameters are enhanced by ex vivo receptor occupancy data. Synthesis and biological evaluation of a novel and highly selective radiolabeled CB1 antagonist is described. The radioligand was used to conduct ex vivo receptor occupancy studies., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The cannabinoid CB(1) G protein-coupled receptor has been shown to be a regulator of food consumption and has been studied extensively as a drug target for the treatment of obesity. To advance understanding of the receptor's three-dimensional structure, we performed mutagenesis studies at human cannabinoid CB(1) receptor residues F200 and S383 and measured changes in activity and binding affinity of compounds from two recently discovered active chemotypes, arylsulfonamide agonists and tetrahydroquinoline-based inverse agonists, as well as literature compounds. Our results add support to previous findings that both agonists and inverse agonists show varied patterns of binding at the two mutated residue sites, suggesting multiple subsites for binding to the cannabinoid CB(1) receptor for both functional types of ligands. We additionally find that an F200L mutation in the receptor largely restores binding affinity to ligands and significantly decreases constitutive activity when compared to F200A, resulting in a receptor phenotype that is closer to the wild-type receptor. The results downplay the importance of aromatic stacking interactions at F200 and suggest that a bulky hydrophobic contact is largely sufficient to provide significant receptor function and binding affinity to cannabinoid CB(1) receptor ligands., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Agonists of the 5-HT(2C) receptor have been shown to suppress appetite and reduce body weight in animal models as well as in humans. However, agonism of the related 5-HT(2B) receptor has been associated with valvular heart disease. Synthesis and biological evaluation of a series of novel and highly selective dihydroquinazolinone-derived 5-HT(2C) agonists with no detectable agonism of the 5-HT(2B) receptor is described. Among these, compounds (+)-2a and (+)-3c were identified as potent and highly selective agonists which exhibited weight loss in a rat model upon oral dosing., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on side chain substitution. Typical activities of the most potent compounds in this class are in the low double-digit to single-digit nanomolar range in in vitro assays using human ACC1 and ACC2 enzymes.

Acyl coenzyme A:diacylglycerol acyltransferase 1 (DGAT1) is one of the four intestinal membrane bound acyltransferases implicated in dietary fat absorption. Recently, it was found that, in addition to acylating diacylglycerol (DAG), DGAT1 also possesses robust enzymatic activity for acylating monoacylglycerol (MAG) (Yen, C. L., Monetti, M., Burri, B. J., and Farese, R. V., Jr. (2005) J. Lipid Res. 46, 1502-1511). In the current paper, we have conducted a detailed characterization of this reaction in test tube, intact cell culture, and animal models. Enzymatically, we found that triacylglycerol (TAG) synthesis from MAG by DGAT1 does not behave according to classic Michaelis-Menten kinetics. At low concentrations of 2-MAG (<50 microm), the major acylation product by DGAT1 was TAG; however, increased concentrations of 2-MAG (50-200 microm) resulted in decreased TAG formation. This unique product/substrate relationship is similar to MGAT3 but distinct from DGAT2 and MGAT2. We have also found that XP620 is an inhibitor that selectively inhibits the acylation of MAG by DGAT1 (IC(50) of human DGAT1: 16.6+/-4.0 nM (MAG as substrate) and 1499+/-318 nM (DAG as substrate); IC(50) values of human DGAT2, MGAT2, and MGAT3 are >30,000 nM). Using this pharmacological tool, we have shown that approximately 76 and approximately 89% of the in vitro TAG synthesis initiated from MAG is mediated by DGAT1 in Caco-2 cell and rat intestinal mucosal membranes, respectively. When applied to intact cultured cells, XP620 substantially decreased but did not abolish apoB secretion in differentiated Caco-2 cells. It also decreased TAG and DAG syntheses in primary enterocytes. Last, when delivered orally to rats, XP620 decreased absorption of orally administered lipids by approximately 50%. Based on these data, we conclude that the acylation of acylglycerols by DGAT1 is important for dietary fat absorption in the intestine.

Structure-activity relationships for a series of pyrazine carboxamide CB1 antagonists are reported. Pharmaceutical properties of the series are improved via inclusion of hydroxyl-containing sidechains. This structural modification sufficiently improved ADME properties of an orally inactive series such that food intake reduction was achieved in rat feeding models. Compound 35 elicits a 46% reduction in food intake in ad libidum fed rats 4-h post-dose.

Robust pharmaceutical treatment of obesity has been limited by the undesirable side-effect profile of currently marketed therapies. This paper describes the synthesis and optimization of a new class of pyrazinoisoindolone-containing, selective 5-HT2C agonists as antiobesity agents. Key to optimization of the pyrazinoisoindolone core was the identification of the appropriate substitution pattern and functional groups which led to the discovery of (R)-9-ethyl-1,3,4,10b-tetrahydro-7-trifluoromethylpyrazino[2,1-a]isoindol-6(2H)-one (58), a 5-HT2C agonist with >300-fold functional selectivity over 5-HT2B and >70-fold functional selectivity over 5-HT2A. Oral dosing of 58 reduced food intake in an acute rat feeding model, which could be completely reversed by a selective 5-HT2C antagonist and caused a reduction in body weight gain in a 4-day rat model.

Indiplon (NBI 34060; N-methyl-N-[3-[3-(2-thienylcarbonyl)-pyrazolo[1,5-alpha]pyrimidin-7-yl]phenyl]acetamide), a novel pyrazolopyrimidine and high-affinity allosteric potentiator of GABA(A) receptor function, was profiled for its effects in rodents after oral administration. In mice, indiplon inhibited locomotor activity (ED(50) = 2.7 mg/kg p.o.) at doses lower than the nonbenzodiazepine hypnotics zolpidem (ED(50) = 6.1 mg/kg p.o.) and zaleplon (ED(50) = 24.6 mg/kg p.o.), a sedative effect that was reversed by the benzodiazepine site antagonist flumazenil. Indiplon inhibited retention in the mouse passive avoidance paradigm over a dose range and with a temporal profile that coincided with its sedative activity. Indiplon, zolpidem, and zaleplon were equally effective in inhibiting locomotor activity in the rat and produced dose-related deficits on the rotarod. In a rat vigilance paradigm, indiplon, zolpidem, and zaleplon produced performance deficits over a dose range consistent with their sedative effects, although indiplon alone showed no significant increase in response latency. Indiplon produced a small deficit in the delayed nonmatch to sample paradigm at a dose where sedative effects became apparent. Indiplon was active in the rat Vogel test of anxiety, but it showed only a sedative profile in the mouse open field test. The pharmacokinetic profile of indiplon in both rat and mouse was consistent with its pharmacodynamic properties and indicated a rapid T(max), short t(1/2), and excellent blood-brain barrier penetration. Therefore, indiplon has the in vivo profile of an efficacious sedative-hypnotic, in agreement with its in vitro receptor pharmacology as a high-affinity allosteric potentiator of GABA(A) receptor function, with selectivity for alpha1 subunit-containing GABA(A) receptors.

In the last decade, the G-Protein-Coupled Receptor (GPCR) superfamily has emerged as a very promising and enriched source of therapeutic targets for the treatment of obesity. GPCRs represent the largest family of mammalian proteins, with approximately 1000 members. It is estimated that the GPCR family may comprise greater than 1% of the human genome and is the molecular target for approximately 30% of currently marketed drugs. Human GPCRs are modulated by a large variety of ligands, including peptides, lipids, neurotransmitters, nucleotides, ions and external sensory signals such as pheromones, tastes or odors. Many of the above ligands have been implicated in the physiological control of energy balance. This article will examine the biological rationale, assets, identified liabilities and current drug development status of these receptors as anti-obesity drug targets.

We compared the in vivo efficacy of two selective CRF2 agonists, mouse urocortin II (mUcn II) and human urocortin III (hUcn III), using food intake, anxious behavior, or ACTH release in CD-1 or Balb/c mice as indices of biological stress responses. All three peptides produced anorexia (Minimal Effective Dose (M.E.D.) for CRF and mUcn II = 0.03 nmol; M.E.D. for hUcn III = 0.3 nmol). Only mUcn II and CRF appeared to increase anxious behaviors in the elevated plus maze test (M.E.D. = 0.3 and 0.01 nmol, respectively). CRF increased the release of plasma ACTH (M.E.D. of 0.3 nmol), while mUcn II and hUcn III had no effect on ACTH release. These data suggest that the CRF2 receptor subtype plays a primary role in the activation of behavioral, but not neuroendocrine, stress responses., (Copyright 2004 Elsevier Inc.)

Rationale: Hypersecretion of corticotropin releasing factor (CRF) has been implicated in both severe anxiety disorders and major depression. Although the role of the CRF1 receptor in the anxiogenic effects of CRF is well supported, the role of CRF2 receptors in anxiety-like behaviors is less clear. In rats, CRF increases the acoustic startle reflex (ASR) via its action in the extended amygdala, providing a putative measure of CRF-mediated anxiogenic activity., Objective: To characterize the effect of CRF on ASR in mice and determine the respective roles of CRF1 and CRF2 receptors in CRF-potentiated ASR., Methods: The present study examined: (1) the time course and dose response functions for the effects of human/rat (h/r)-CRF (0.02-0.6 nmol, ICV (intracerebroventricular)) on ASR in two inbred strains of mice; (2) the effects of the CRF1 receptor antagonist NBI-30775 (20 mg/kg, intraperitoneal) and the CRF2 receptor antagonist Antisauvagine-30 (1-10 nmol, ICV) on CRF-potentiated ASR and (3) the effects of the CRF2 receptor agonist urocortin 2 (0.2-6 nmol, ICV) on ASR in mice., Results: h/r-CRF significantly increased ASR in mice in a time-dependent manner with maximal efficacy at the 0.2 and 0.6 nmol doses. 129S6/SvEvTac mice exhibited a slightly increased duration of action and lower minimal effective dose threshold for CRF effects on ASR compared to C57BL/6J mice. Both selective CRF1)and CRF2)antagonists attenuated h/r-CRF-potentiated ASR without affecting acoustic startle when given alone. The selective CRF2 receptor agonist urocortin 2 increased ASR (1 and 2 nmol), albeit with less efficacy than the non-selective CRF receptor agonist h/r-CRF., Conclusions: Both CRF1 and CRF2 receptors appear to contribute to the h/r-CRF-induced increases in ASR in mice. These data support the hypothesis that both receptors contribute to the anxiogenic effects of CRF.

Energy balance is a highly regulated, complex process which is modulated by central and peripheral systems. Dysregulation of energy homeostasis can result in metabolic disorders, such as obesity and type II diabetes. Obesity and type II diabetes are two of the most prevalent and challenging clinical conditions in society today. A growing body of evidence has implicated the melanocortin system as an important component in the maintenance of energy balance. alpha-MSH, a 13 amino acid peptide secreted as a product of the pro-opiomelanocortin (POMC) gene in the pituitary is a potent agonist of 4 of the 5 cloned melanocortin receptors (MCR). MC receptors are members of a G-protein-coupled receptor (GPCR) family, which signal through cAMP. Agouti and agouti-related protein (AGRP) are natural antagonists of melanocortin receptors and participate in regulation of skin/fur pigmentation, body weight, and adiposity. Stimulation of MC receptors has pleiotropic effects, which impact the nervous system as well as endocrine and immune functions. One of the most prominent effects of MC receptor stimulation is a dramatic suppression of food intake and body weight, which has led to the hypothesis that the MC receptor system plays a primary role in the maintenance of energy balance. This idea is supported by a large body of pharmacological, molecular and human genetic evidence. The following review summarizes the role of melanocortin receptors in the regulation of food intake and energy homeostasis and highlights the opportunities for MC receptors as drug development targets in treating eating disorders and diabetes.

Corticotropin-releasing factor (CRF) is one of the principle components of the stress response. The physiological effects of CRF are mediated by two receptor subtypes, CRF(1) and CRF(2). Recent data obtained with the selective CRF(2) antagonist antisauvagine-30 (ASV-30) has begun to suggest that both CRF receptor subtypes may play a role in stress-related behaviors. Exactly how these two receptor subtypes interact to modulate the behavioral and endocrine responses to stress is not clear, however. We have attempted to understand the role of the CRF(2) receptor in the behavioral and endocrine responses to stress by comparing the effects of ASV-30 with the mixed CRF(1)/CRF(2) receptor antagonist astressin. Centrally administered ASV-30 reduced anxiety-like behavior in BALB/c mice in three models of anxiety: marble burying [minimal effective dose (MED) = 3 nmol], open field (MED = 3 nmol), and elevated plus maze (MED = 0.1 nmol). ASV-30 did not change locomotor activity or the adrenocorticotropic hormone (ACTH) response to restraint stress. The potent mixed CRF(1)/CRF(2) antagonist astressin not only reduced anxiety-like behavior in all three models with equivalent potency but also blunted the ACTH response to restraint stress. Finally, the new selective CRF(2) receptor agonist urocortin-II produced a dose-dependent increase in anxiety-like behavior in the plus maze test. Therefore, our data suggest that the CRF(2) receptor plays a role in the behavioral, but not the hypothalamic-pituitary-adrenal axis, response to stress.

The two protocols in this unit provide suggestions for constructing models of eating disorders that are at the opposite ends of the spectrum: dietary hyperphagia and anorexia nervosa. The greatest degree of dietary hyperphagia is induced by giving rats or mice access to a daily choice of highly palatable foods (e.g., chocolate or bread) in addition to their regular chow. Like humans, rats overeat and actually develop physiological requirements for these foods. This model can be used to test the effects of putative anorectic agents on both acute and chronic administration regimens. The second protocol describes a model of compulsive behavior that results in profound weight loss, which is produced by moderate food deprivation along with continuous access to exercise wheels.

Although there is considerable information regarding the role of brain CRF in energy balance, relatively little is known about the role of urocortin (UCN), which is an equally potent anorexic agent. Therefore, the effects of intracerebroventricular (icv) administration of UCN (0.01-1 nmol/day) on food intake and body weight were assessed over a period of 13 days and compared with data from CRF-infused counterparts. Although both peptides dose dependently reduced food intake and weight gain, the effects of CRF were much greater in magnitude than those of UCN, particularly on body weight. Pair-feeding studies suggested that, while the effects of CRF on body weight could not be completely explained by appetite suppression, the effects of UCN appeared to be due to its initial impact on food intake. CRF increased brown adipose fat pad and adrenal weights, whereas it reduced thymus and spleen weights. CRF also increased serum corticosterone, triglyceride, FFA, and cholesterol levels, whereas it reduced glucose. UCN did not produce any consistent changes in any of these indices of sympathetic nervous system activation. Concurrent administration of the CRF(2)-selective antagonist, antisauvagine-30 (ASV-30) (30 nmol/day) completely reversed or attenuated the effects of UCN and CRF (1 nmol/day) on food intake and body weight. ASV-30 did not significantly attenuate any of the above CRF-induced changes in tissue weights or serum chemistry. These data suggest that the central CRF(2) receptor may primarily mediate the anorexic, but not the metabolic effects of CRF.

The central melanocortin system is critical for the long term regulation of energy homeostasis. Null mutations of the melanocortin-4 receptor (MC4-R) are associated with hyperphagia, obesity, and accelerated longitudinal growth in mice and humans. However, little is known about the function of another central melanocortin receptor, the MC3-R. To assess the role of the MC3-R in energy homeostasis, the majority of the mc3r coding sequence was deleted from the mouse genome. In contrast to the MC4-R knockout, which exhibits increased food intake, increased somatic growth, and defects in metabolism, mc3r-/- mice exhibit an exclusively metabolic syndrome. Homozygous null mc3r mice, while not significantly overweight, exhibit an approximately 50% to 60% increase in adipose mass. Mc3r-/- mice also exhibit an unusual increase in respiratory quotient when transferred onto high fat chow, suggesting a reduced ratio of fat/carbohydrate oxidation. Furthermore, male mc3r-/- mice also exhibit an approximately 50% reduction in locomotory behavior on the running wheel, suggesting reduced energy expenditure.

Genetic manipulations of corticotropin-releasing factor (CRF)(1) and CRF(2) receptors have resulted in data suggesting that the CRF(2) receptor could mediate the effects of CRF on appetite or satiety. We have attempted to obtain pharmacological evidence for this hypothesis by comparing the ability of a high-affinity peptide, mixed CRF antagonist [cyclo 30-33,f12,L18,21E30, A32,K33]sucker fish urotensin (12-41)NH(2) [cUTSN (12-41)] with a small-molecule CRF(1)-selective antagonist, NBI-27914, and a CRF(2)-selective peptide antagonist, antisauvagine-30, to attenuate the anorexic effects of CRF. We also monitored other behaviors that accompanied CRF-induced anorexia. CRF-induced anorexia was significantly correlated with a reduction in locomotor activity and an increase in freezing behavior and piloerection. cUTSN (12-41) and antisauvagine-30 significantly attenuated the effects of CRF (0.04 nmol) on food intake along with the behavioral syndrome that accompanied anorexia. In contrast, NBI-27914 did not attenuate either of the above-mentioned CRF-induced phenomena when given centrally at doses ranging from 0.13 to 10 nmol/2.5 microl or when given orally at 20 to 40 mg/kg. Although these data support the hypothesis that the CRF(2) receptor mediates the appetite suppression induced by CRF, they also suggest that the CRF(2) receptor could mediate the stress-like behaviors that accompany CRF-induced appetite suppression.

Leptin is a 16 kD protein that is produced by adipocytes and induces weight loss in both normal and genetically obese ob/ob mice. ob/ob mice are obese, have multiple metabolic abnormalities, and exhibit impaired wound healing. Exogenous administration of leptin to these animals induces weight loss and corrects their metabolic defects. Leptin's effect on wound repair, however, has not been studied. Systemic administration of leptin at doses ranging from 0.1 to 10 mg/kg/day induced a highly significant acceleration in wound repair in ob/ob mice (p<0.0001), but not in db/db mice, indicating that leptin's effects on wound repair were mediated through the leptin receptor. We then investigated the possibility that leptin was acting directly at the wound site by administering leptin topically, and found that topical leptin also induced a dose dependent acceleration in wound repair (p<0.0001). In addition, we found that all forms of leptin receptor, including the signal transducing long form, were present in skin by RNase protection assay, and that leptin receptor localized to subcutaneous vessels of wounded skin by in situ hybridization. Finally, we investigated the possibility that leptin stimulated angiogenesis in wounds by analyzing wound hemoglobin and wound vessel density. Neither systemic nor topical leptin induced any significant changes in either parameter, suggesting that leptin accelerates wound repair by a mechanism other than stimulation of angiogenesis. In summary, both systemic and topical leptin accelerate wound repair in diabetic ob/ob mice, possibly via the direct interaction of leptin with its receptors in wounded skin, but do not appear to significantly stimulate wound angiogenesis. Further studies to better elucidate the mechanisms of leptin's effects on wound repair are warranted.

To characterize age-related changes in frontal cortical plasticity, we assessed maze learning and frontal cortical pharmacology in young adult, middle-aged, and aged rats. Rats received either ibotenic acid or sham lesions of the nucleus basalis magnocellularis (NBM) and were then trained on a radial maze task. After training, we assessed [3H]desmethylimipramine (DMI), [3H]muscimol, [3H]AMPA, and [3H]QNB binding using quantitative autoradiography. Both middle-aged and aged rats were impaired on the radial maze task. DMI binding was increased in both middle-aged and aged rats, while QNB binding was decreased in aged rats. While lesions impaired maze performance at all ages, middle-aged and aged rats showed more profound lesion-induced deficits. Lesions increased GABA, and AMPA receptor binding in young adult rats only. These lesion-induced changes may reflect a compensatory response that is lost with advancing age.

The role of estradiol in mediating leptin's effects on body weight was assessed in ovariectomized (OVX) mice before and after the onset of obesity. Ovariectomy did not alter leptin levels before the onset of obesity, and estradiol adminstration (0.05-17 microgram/day for 14 days) did not significantly alter leptin levels if they were corrected for the estradiol-induced reduction in body fat. The converse was also true, in that leptin administration (0.4-140 microgram/day) did not alter estradiol levels in intact mice. Furthermore, neither estradiol reduction (via ovariectomy) nor addition (via exogenous administration) significantly altered leptin's ability to reduce fat mass. Leptin was equally effective in reducing body weight in lean or obese OVX mice and intact controls. Finally, estradiol did not change the magnitude of leptin's effect on fat mass reduction when it was given in combination with leptin to lean intact or OVX mice. Estradiol may have indirectly affected leptin efficacy, because leptin did not produce as large a change in fat mass at lower doses in lean OVX mice as it did in intact counterparts. Taken together, these data suggested that 1) estradiol does not directly regulate leptin secretion or its effects on fat mass and 2) leptin does not directly regulate estradiol secretion or its effects on fat mass. Leptin and estradiol, however, may interact in an indirect fashion to affect fat utilization.

Taurine is present in high concentration in the mammalian brain and is known to decline with aging. The present studies examined the relationship between the loss of striatal neurotransmitters and spatial learning ability in aged male Long-Evans rats. The effects of intrahippocampal infusions of neurotrophic factors-nerve growth factor (NGF) and brain-derived neurotrophic factor-were also examined for their ability to ameliorate the age-related decline in brain amino acid content. Taurine content was found to be significantly reduced in the striatum of aged rats (26 months old) that were impaired in spatial learning performance when compared to young unimpaired rats (5 months old). Aged rats that were behaviorally unimpaired had more modest reductions in taurine. Striatal dopamine content was also significantly reduced in aged learning-impaired rats. There was a significant (p < 0.001) correlation (r=0.61) between the striatal content of taurine and dopamine, but no such correlation was found for other striatal transmitters (glutamate, serotonin, norepinephrine). Treatment with neurotrophins had little effect on the age-related decline in striatal amino acids, although NGF treatment did improve spatial learning. These studies suggest (1) a link between age-related declines in striatal dopamine and taurine and (2) that NGF-induced improvement in spatial learning is not related to mechanisms involving changes in taurine or glutamate content.

Leptin efficacy was compared in obese and lean female CD-1 mice. Body weights in these 10- to 12-mo-old mice ranged from 29.7 to 62.0 g, and leptin levels correlated with body weight. Mice from the lean and obese ends of the weight distribution were treated with daily peripheral leptin injections (1-100 mg/kg) for a 33-day period. The half-maximal effective doses for weight loss and fat reduction were shifted 0.5-0.7 log to the right for obese mice. Leptin was less efficacious at low doses (1-3 mg/kg) in obese mice but equal to or more efficacious in obese than lean mice at high doses (30-100 mg/kg). Leptin's initial effects on weight loss could be explained by appetite suppression in both groups, but its effects on fat reduction were greater in leptin-treated than pair-fed mice, particularly in the lean group. Leptin also prevented the elevations in serum corticosterone and ketones found in pair-fed lean mice. These data allow a quantitative comparison of leptin sensitivity in obese vs. lean CD-1 mice and suggest that in mice where obesity is a function of outbreeding and age, leptin sensitivity is moderately reduced. Furthermore, although appetite suppression has a clear role in leptin's effects on body weight, leptin may also have specific effects on lipid metabolism and mobilization that are different from the metabolic compensations that normally occur with food deprivation.

In mammals, leptin reduces energy intake and may increase energy expenditure as a means to maintain body weight and/or adiposity at an appropriate level. Hibernating mammals seasonally alter body mass, food intake, and body composition and, therefore, represent an attractive model for investigating the physiological regulation of changing body mass and adiposity. Previous experiments in our laboratory demonstrated that administration of mouse recombinant leptin reduces food intake and body weight in arctic ground squirrels during prehibernation fattening. In addition, leptin appeared to reduce metabolic efficiency (weight gain per unit of energy intake). This result suggests that reduced food intake alone may not account for the observed weight loss. Here, we describe the effect of a 3-week constant infusion of leptin given to posthibernation arctic ground squirrels on food consumption and energy expenditure. Mouse recombinant leptin (1 mg/ml) was administered through subcutaneously implanted mini-osmotic pumps (10 microliters/hr flow rate). Resting metabolic rate was monitored before and during the 3-week leptin administration period by indirect calorimetry. Body temperature and locomotory activity were monitored continuously by abdominal radiotransmitters. At the end of the leptin administration period, thermogenic capacity was evaluated by measuring brown fat uncoupling protein-1 mRNA and protein levels. Leptin administration resulted in reduced food intake and prevented posthibernation weight gain, but it did not alter any of the measured parameters of energy expenditure.

Leptin is a protein secreted by adipocytes that is important in regulating appetite and adiposity. Recent studies have suggested the presence of leptin receptors in the arcuate nucleus of the hypothalamus (ANH). Neonatal administration of monosodium glutamate (MSG) damages the ANH, resulting in obesity and neuroendocrine dysfunction. Neonatal administration of MSG was utilized to test the hypothesis that the anatomic site for many of leptin's actions is the ANH. Female control (n = 6) and MSG-treated rats (n = 7) were implanted for 14 days with osmotic minipumps containing phosphate-buffered saline or leptin (1 mg.kg-1.day-1). Leptin suppressed (P < 0.05) body weight gain in controls but did not suppress weight gain in MSG-treated rats. Leptin decreased (P < 0.05) fat depots in controls but had no effect in MSG-treated rats. Night feeding was suppressed (P < 0.05) in leptin-treated control rats. MSG-treated rats showed a suppression in food intake that was of a smaller magnitude and appeared later in the course of leptin treatment. These findings suggest that leptin mediates some physiological actions related to fat mobilization via receptors located in the ANH.

The ob gene product leptin is thought to play a physiological role in the fine tuning of a homeostatic mechanism regulating satiety and adiposity. Mouse recombinant leptin was administered to seasonally hyperphagic arctic ground squirrels as a first step in demonstrating the evolutionary conservation of leptin function and the potential involvement of leptin in the seasonal regulation of adiposity in hibernators. Continuous infusion of leptin for 3 wk via miniosmotic pumps resulted in a reduction in food intake and body weight in a manner consistent with its proposed role as a satiety hormone. During the recovery period after leptin administration, squirrels that had received leptin became hyperphagic relative to controls. Percent body fat was estimated at weekly intervals by measuring total body electrical conductivity and decreased after 3 wk of leptin administration. Our observations support the role of leptin as a regulatory hormone involved in the control of satiety, adiposity, and possibly energy expenditure in hibernating mammals.

Spatial learning rate was compared in cognitively impaired aged rats infused with either brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF). BDNF or NGF was infused into the dorsal hippocampus/third ventricle while animals were being trained on the Morris water maze. Training continued until all rats met a spatial learning criterion. Seven weeks later, they were tested for retention of the task, and sacrificed for assessment of hippocampal high-affinity choline uptake (HACU) or hypothalamic biogenic amine levels. NGF, but not BDNF, improved spatial learning rate in aged rats and increased hippocampal choline uptake weeks after withdrawal of NGF. Although BDNF did not improve spatial learning, it did induce a partial, long-term normalization of the elevated hypothalamic 5-HT levels observed in our aged rats. These data suggest that (1) intrahippocampal/intraventricular infusion of NGF can improve the learning rate of aged, spatial learning-impaired rats, and that this improvement in acquisition could be associated with increased hippocampal cholinergic activity, and (2) that the BDNF-induced normalization of hypothalamic 5-HT levels in aged rats was not sufficient to improve learning rate in aged, spatial learning-impaired rats.

Glial cell line-derived neurotrophic factor (GDNF) was infused continuously for 2 weeks into the ventricles of male Wistar rats that had received a unilateral knife transection of the fimbria/fornix. In vehicle-treated, control animals, there was a 70% loss of choline acetyltransferase (ChAT)-positive and a 60% loss of p75-positive neurons in the septum/diagonal band ipsilateral to the axotomy as identified by immunohistochemistry, with no loss in ChAT biochemical activity. GDNF treatment at 10 micrograms/day completely prevented the loss of p75-positive neurons, significantly reduced the loss of ChAT-positive neurons to 40% of normal, and stimulated ChAT biochemical activity to 40% more than normal in an axotomy-dependent manner. GDNF is 1 order of magnitude less potent than NGF but, unlike NGF, had little or no effect on normal, uninjured neurons. GDNF was 1 order of magnitude more potent than BDNF, and BDNF had no effect on ChAT biochemical activity. GDNF and NGF inhibited weight gain, whereas BDNF induced significant weight loss and death at the dosage of 100 micrograms/day.

The hypothalamus plays a central role in the integrated regulation of energy homeostasis and body weight, and a number of hypothalamic neuropeptides, such as neuropeptide Y (ref. 1), galanin, CRH (ref. 3) and GLP-1 (ref. 4), have been implicated in the mediation of these effects. To discover new hypothalmic peptides involved in the regulation of body weight, we used differential display polymerase chain reaction to identify messenger RNAs that are differentially expressed in the hypothalamus of ob/+ compared with ob/ob C57B1/6J mice. We show here that one mRNA that is overexpressed in the hypothalamus of ob/ob mice encodes the neuropeptide melanin-concentrating hormone (MCH). Fasting further increased expression of MCH mRNA in both normal and obese animals. Neurons containing MCH are located in the zona incerta and in the lateral hypothalamus. These areas are involved in regulation of ingestive behaviour, but the role of MCH in mammalian physiology is unknown. To determine whether MCH is involved in the regulation of feeding, we injected MCH into the lateral ventricles of rats and found that their food consumption increased. These findings suggest that MCH participates in the hypothalamic regulation of body weight.

Neuropeptide Y (NPY), a 36-residue polypeptide produced abundantly in both nervous and peripheral tissues, appears to play a significant role in the regulation of diverse biological processes, including feeding behavior and cardiovascular and psychotropic functions. The actions of NPY are mediated through effective binding to specific receptors of which two, designated Y1 and Y2, have been well characterized. A shortened cyclic analogue of NPY, des-AA10-17-cyclo-7/21[Cys7,21]NPY, was shown to retain high affinity for both human neuroblastoma SK-N-MC and SK-N-BE2 cell types (expressing Y1 and Y2 receptors, respectively). Increasing the size of the ring (des-AA10-17-cyclo-2/27[Cys2,27]NPY) in the present study produced a high-affinity analogue (Ki = 3.0 vs 0.3 nM for NPY) that bound exclusively to Y2 receptors. Using the feedback from structure-activity relationships, we also describe the optimization of specific substitutions and bridging arrangements leading to the production of other truncated, high-affinity Y1 selective analogues which bind, as does NPY itself, in the low-nanomolar range. Of greatest significance, des-AA10-17-cyclo-7/21[Cys7,21,Pro34]NPY (11) was found to possess agonistic properties with an affinity comparable to that of the native NPY molecule when tested for its ability to inhibit norepinephrine-stimulated cAMP release in SK-N-MC human neuroblastoma cells. Compound 11 also caused an increase in blood pressure in anesthetized rats. However, in two central nervous system models of Y1 receptor function, stimulation of feeding and anxiolytic activity, this analogue was inactive, which suggests the presence of a new subclass of receptors. In summary, the present results demonstrate that residues 10-17 of NPY are not directly involved in either Y1 or Y2 receptor recognition or activation. This suggests that the selectivity of NPY receptors is highly dependent on subtle conformational changes such as the substitution of residue 34 to a proline or the introduction of intramolecular constraints. Additionally, we have produced an analogue of NPY that selectively activates peripheral NPY Y1 receptors.

C57BL/6J mice with a mutation in the obese (ob) gene are obese, diabetic, and exhibit reduced activity, metabolism, and body temperature. Daily intraperitoneal injection of these mice with recombinant OB protein lowered their body weight, percent body fat, food intake, and serum concentrations of glucose and insulin. In addition, metabolic rate, body temperature, and activity levels were increased by this treatment. None of these parameters was altered beyond the level observed in lean controls, suggesting that the OB protein normalized the metabolic status of the ob/ob mice. Lean animals injected with OB protein maintained a smaller weight loss throughout the 28-day study and showed no changes in any of the metabolic parameters. These data suggest that the OB protein regulates body weight and fat deposition through effects on metabolism and appetite.

There is evidence that central infusion of brain-derived neurotrophic factor (BDNF) induces weight loss in rats. We have begun to investigate the physiological basis for BDNF-induced weight loss by assessing its relationship to (a) appetite, (b) serum indices of metabolic and renal toxicity, and (c) brain monoamine activity in areas associated with feeding or motor function. BDNF (0-6 microgram/day) was infused into the lateral ventricle (LV) of male Long-Evans rats for 14 days. Body weight and food intake were monitored throughout infusion and recovery periods. BDNF induced severe, dose-dependent appetite suppression and weight loss. Although appetite began to recover after the 10th infusion day, body weight had not returned to control values at the end of the recovery period. The weight loss observed in BDNF-infused rats was related to appetite suppression, since uninfused rats that were pair-fed to high dose BDNF-treated rats showed comparable weight loss. Despite severe weight loss, serum BUN, creatinine, thyroxine, glucose, and total protein were not affected by BDNF infusion. Striatal DO-PAC/DA was similarly unaffected by BDNF. In contrast, BDNF-infused rats showed a dose-dependent increase in hypothalamic 5-HIAA/5-HT that was not observed in pair-fed rats, suggesting that the observed increase in hypothalamic 5-HIAA/5-HT was a direct effect of BDNF infusion rather than a secondary effect of food restriction. These data suggest that BDNF may induce appetite suppression and weight loss through a central mechanism.

Idebenone (6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone) is a benzoquinone that has been shown to improve cognitive function in animals subjected to cerebral ischemia and in rats with lesions of the basal forebrain cholinergic system. Because the cognitive deficits observed in aged rats have been associated with decreased cerebral blood flow and basal forebrain cholinergic dysfunction, it was hypothesized that IDE might improve cognition in aged animals. In the present study, the effects of idebenone on cognitive function in aged Long-Evans rats were assessed using a battery of tests that evaluated attention, habituation, and spatial learning. Selective attention was assessed using an overshadowing paradigm, where IDE (30 mg/kg, IP) was injected 30 min prior to compound cue exposure. IDE enhanced the overshadowing effect in aged rats. The Morris water maze was used to assess spatial learning, where IDE (3 mg/kg, IP) was injected daily throughout the course of training. IDE did not improve the impaired performance of aged rats in the Morris task. Habituation was tested by measuring recovery from gustatory neophobia. IDE (30 mg/kg, IP) was injected 30 min prior to the first exposure to the novel taste. IDE normalized habituation rate in aged rats. It was concluded that IDE improves some forms of acquisition in aged rats, and may do so by decreasing general reactivity to novel stimuli.